US5753787A - Nucleic acids encoding ancylostoma secreted protein - Google Patents
Nucleic acids encoding ancylostoma secreted protein Download PDFInfo
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- US5753787A US5753787A US08/419,414 US41941495A US5753787A US 5753787 A US5753787 A US 5753787A US 41941495 A US41941495 A US 41941495A US 5753787 A US5753787 A US 5753787A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention is generally in the area of treatments and diagnostics for hookworm infection.
- Hookworm disease continues to rank among the most important infectious diseases on a world-wide basis. Over 20% of the world population is infected, and hookworms are the leading cause of anemia in the tropics (Gilles, Reviews of Infectious Diseases 7: 111-118 (1985)). In China alone, an estimated 194 million people are infected with hookworm, with up to 50 percent prevalence in the agriculturally rich areas along the Yangtze River.
- hookworm remains a serious geohelminth infection. This is due to several factors. First, many of the latest generation of anthelminthics are prohibitively expensive, and therefore unaffordable by the developing countries that would benefit most from their use. Secondly, there is no sterile immunity developed following hookworm infection, such that people treated with anthelminthics are quickly re-infected when they are re-exposed to infective stages. Third, previously unreported toxicities have been now observed with wide scale use of anthelminthic drugs including seizures, encephalitis and deafness. Fourth, disappointing efficacy has been noted with the conventional anthleminthic drugs.
- An effective and inexpensive recombinant vaccine would provide an affordable approach to controlling the ravages of hookworm disease.
- the protein has been designated Ancylostoma Secreted Protein (ASP).
- ASP has an apparent molecular weight of 37-40 kDa.
- ASP from Ancylostoma caninum contains 406 amino acids.
- the pro-form of ASP contains an 18 amino acid secretion signal sequence which does not appear in mature ASP.
- ASP is released from larval A. caninum that begin feeding.
- DNA encoding the protein has been isolated.
- the protein can be made by recombinant means in a variety of hosts.
- the recombinant protein, or immunogenic fragments thereof, can be formulated as a vaccine.
- the recombinant protein, or antibodies to the protein, can also be used in an assay to detect hookworm infection.
- DNA encoding all or parts of the protein can be used as probes to detect the presence of hookworm nucleic acid. Such probes are useful in assays of tissue samples, body fluid samples, and hookworm infection sources.
- a protein that is released by hookworm larvae following infection designated Ancylostoma Secreted Protein.
- the protein has an apparent molecular weight of 37-40 kDa.
- ASP from Ancylostoma caninum contains 406 amino acids.
- the pro-form of ASP contains an 18 amino acid secretion signal sequence which does not appear in mature ASP.
- the sequence of A. caninum ASP is listed as SEQ ID NO:2. This sequence includes the 18 amino acid secretion signal sequence.
- ASP is released from larval A. caninum that begin feeding. Larvae begin feeding at the transition from the free-living stage to the parasitic stage. ASP is thought to be involved in this transition.
- Ancylostoma Secreted Protein and ASP refer to a protein having the amino acid sequence shown in SEQ ID NO:2 from amino acid 19 to amino acid 424; derivatives thereof with conservative amino acid substitutions, deletions, or additions; any homolog or allelic form of ASP produced in nematodes; any protein having at least 40% amino acid sequence identity with amino acids 19 to 424 of SEQ ID NO:2; or any protein that is immunoreactive with an antibody immunoreactive with the ASP of SEQ ID NO:2 but that is not immunoreactive with hymenoptera proteins.
- ASP derivatives have an amino acid sequence identity with amino acids 19 to 424 of SEQ ID NO:2 of greater than 40%, such as at least 50% amino acid sequence identity, at least 60% amino acid sequence identity, or at least 70% amino acid sequence identity.
- ASP derivatives have at least 80% amino acid sequence identity; more preferably, at least 90% amino acid sequence identity; and most preferably, at least 95% amino acid sequence identity with amino acids 19 to 424 of SEQ ID NO:2.
- percent amino acid sequence identity is calculated as the percentage of aligned amino acids that match the reference sequence, where the sequence alignment has been determined using the alignment algorithm of Dayhoff et al., Methods in Enzymology 91: 524-545 (1983).
- the reference sequence is amino acids 19 to 424 of SEQ ID NO:2.
- all amino acid position references use the numbering shown in SEQ ID NO:2.
- immunoreactivity is determined by any standard technique for detecting antibody-antigen interactions. Many assays useful for determining immunoreactivity are described in Johnstone and Thorpe, Immunochemistry in Practice, Second edition (Blackwell Scientific Publications, Oxford, 1987). Preferred assays are the immunoassays described on pages 241 to 260 of Johnstone and Thorpe.
- preferred fragments are those that are not immunoreactive with antibodies that are immunoreactive to proteins other than ASP. Accordingly, preferred immunogenic fragments of ASP have an amino acid sequence with at least five consecutive amino acids that are present in an ASP as defined above, where no amino acid sequence of five or more, six or more, seven or more, or eight or more consecutive amino acids present in the immunogenic fragment of ASP is present in a protein other than ASP. More preferred immunogenic fragments of ASP have an amino acid sequence of at least five consecutive amino acids in SEQ ID NO:2, where no amino acid sequence of five or more, six or more, seven or more, or eight or more, consecutive amino acids present in the immunogenic fragment of ASP is present in a protein other than ASP.
- Antibodies against the antigen 5 of the yellow jacket Vespula squarnosa recognize a 40 kDa protein in activated, but not in non-activated, A. caninum L 3 excretory/secretory (ES) products. Additionally, this antibody recognizes a 38 kDa protein in lysates of bacteria that are expressing a one kilobase 3'-end fragment of the ASP cDNA, but not in uninduced bacteria.
- the anti-antigen 5 antibody recognizes bands of slightly higher molecular weight, approximately 42 kDa, in soluble extracts of A. caninum adults and L 3 and A. braziliense, Haemonchus contortus, and Strongyloides stercoralis L 3 . The size difference is due to the presence of the 18 amino acid eukaryotic secretory signal peptide of ASP in the unsecreted form.
- a cDNA molecule encoding ASP has the nucleotide sequence listed as SEQ ID NO:1.
- the coding region in this sequence is 1272 nucleotides long.
- the coding region for mature ASP begins at nucleotide 55 and ends at nucleotide 1272. Unless otherwise stated, all nucleotide position references use the numbering shown in SEQ ID NO:1.
- nucleic acid molecule encoding ASP refers to a nucleic acid molecule encoding a protein having the amino acid sequence shown in SEQ ID NO360:2 from amino acid 19 to amino acid 424; derivatives thereof with conservative amino acid substitutions, deletions, or additions; any homolog or allelic form of ASP produced in nematodes; any protein having at least 40% amino acid sequence identity with amino acids 19 to 424 of SEQ ID NO:2; or any protein that is immunoreactive with an antibody immunoreactive with the ASP of SEQ ID NO:2 but that is not immunoreactive with hymenoptera proteins.
- the encoded ASP derivatives have an amino acid sequence identity with amino acids 19 to 424 of SEQ ID NO:2 of greater than 40%, such as at least 50% amino acid sequence identity, at least 60% amino acid sequence identity, or at least 70% amino acid sequence identity.
- the encoded ASP derivatives have at least 80% amino acid sequence identity; more preferably, at least 90% amino acid sequence identity; and most preferably, at least 95% amino acid sequence identity with amino acids 19 to 424 of SEQ ID NO:2. Amino acid sequence identity and immunoreactivity of the encoded protein are determined as described earlier.
- Nucleic acid molecules encoding all or a part of ASP can be manipulated using any of the numerous nucleic acid manipulation methods known to those of skill in the art. Such manipulation include amplification, purification, recombination, insertion into vectors, and use as probes, consisting of at least 14 to 17 nucleotides, and primers.
- ASP can be made recombinantly using any of the established expression systems, such as bacteria, yeast, baculovirus, and mammalian cell culture.
- recombinant refers to any protein or nucleic acid produced by any methods within the large body of well known nucleic acid manipulations, examples of which are provided below.
- any protein or nucleic acid that results from, or is expressed from a nucleic acid resulting from, such manipulations is a recombinantly produced protein or nucleic acid.
- Recombinant expression allows the production of fragments of ASP, combinations of ASP fragments in a single fusion protein, and fusion proteins combining ASP, or a fragment of ASP, with one or more other proteins or amino acid sequences. Such fusions are useful for combining multiple immunogenic fragments in a single protein.
- the DNA used to produce ASP may be genomic DNA, in which case it may include introns, or it may be cDNA which is prepared in vitro from mRNA using a reverse transcriptase and which contains open reading frames. Methods for isolation, cloning or synthesizing DNA and cDNA are well known to those of skill in the art.
- Expression refers to the process by which nucleic acid is transcribed and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.
- An expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into appropriate host cells, causes nucleic acid molecules that have been cloned into the vector to be transcribed, and then translation of the transcribed nucleic acid into a polypeptide.
- the nucleic acid molecule is cloned into the vector in such a manner that it is operably linked to regulatory sequences that effect expression of the heterologous nucleic acid molecules.
- the expression product may be exported to the cytoplasm and/or may be secreted out of the host cell.
- Appropriate expression vectors are well-known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells. Such expression vectors may remain episomal or may integrate into the host cell genome.
- the ASP cDNA or gene can be inserted into appropriate expression vectors containing expression regulatory elements, such as transcription initiation signals, translation initiation signals, starting codon, termination codon, transcription terminating signals, polyadenylation signals, and others. Suitable vectors are commercially available from a variety of companies. After the recombinant vectors containing ASP-encoding DNA are transfected into the host cells, they may remain as extrachromosomal DNA or they may be integrated into the host genome. In either case, they may direct the synthesis of recombinant ASP in the host cells. Some examples for the expression of heterologous genes are described in Methods in Enzymology, Vol.
- E. coli or other bacteria as host Many mammalian cDNA's have been expressed in E. coli and many expression vectors with different promoters, operators, and other regulatory elements are available commercially. A typical vector construction and expression is described by Lin and Tang, J. Biol. Chem. 264: 4482-4489 (1989). The expression of some eukaryotic proteins in the cytosol of E. coli produces insoluble ⁇ inclusion bodies ⁇ and would require the refolding of recombinant protein. However, the use of a ⁇ leader ⁇ sequence, such as omp, described by Duffaud et al. in Methods in Enzymology 153: 492-506 (Wu and Grossman, eds., Academic Press, 1987), will direct the proper folding and also export of the recombinant ASP to the periplasmic space of the bacterial.
- ⁇ leader ⁇ sequence such as omp
- yeast host cells may express a foreign gene either in the cytosol or as secreted protein.
- Fungi as host There are small numbers of fungal expression vectors which have been successfully used to express heterologous genes.
- the existing fungal expression vectors integrate themselves into the host genome after transfection as indicated by Cullen et al., in A Survey of Molecular Cloning Vectors and their Uses, (Butterworth Publishers, Stoneham, MA 1986).
- the secreted recombinant proteins can be glycosylated.
- Some examples of successful expressions involve bovine chymosin, described by Cullen et al., Bio/Technology 5: 369-378 (1987), and an acid protease from a different fungus, described by Gray et al., Gene 48: 41-53 (1987).
- Insect cells as host Baculovirus expression vectors for the synthesis of foreign genes in insect cells have been successfully used to express many eukaryotic and viral proteins. This system is capable of glycosylation, if the protein has glycosylation sites, and can also express recombinant proteins at a high level. The use of this system has been reviewed in some detail by Luckow and Summers, Bio/Technology, Sep. 11, 1987. The recombinant ASP fusion proteins can also be expressed in insect cells using other expression vectors such as Entomopox viruses and cytoplasmic polyhedrosis viruses (CPV).
- CPV cytoplasmic polyhedrosis viruses
- Mammalian cells as host Many heterologous genes have been expressed in mammalian cells on a commercial scale. The commercial production of recombinant human tissue plasminogen activator is an example. Most of these expression vectors contain 1) either a mammalian promoter, such as metallocyanin or growth hormone, or viral promoters, such as SV40 early promoter or long terminal repeats of viral genes; 2) polyadenylation signals; and 3) appropriate regulatory elements for E. coli cloning including antibiotic resistance genes. After the insertion of ASP downstream from the promoter, the vector can be first cloned in E. coli, isolated and transfected into mammalian cells.
- a mammalian promoter such as metallocyanin or growth hormone
- viral promoters such as SV40 early promoter or long terminal repeats of viral genes
- polyadenylation signals such as SV40 early promoter or long terminal repeats of viral genes
- polyadenylation signals such as SV40 early promoter or long terminal
- Neomycin or similar resistant selection markers can be either cotransfected in another vector or in the same vector.
- a gene amplification system is advantageous.
- the expression vector can contain the gene of dihydrofolate reductase (dhfr).
- dhfr dihydrofolate reductase
- the cloned gene can be coamplified with that of dhfr by adapting the transformed cells to increasing methotrexate concentration.
- the transformant clones secreting ASP can be identified by enzyme assays or by western blots.
- Methods for purifying proteins are well known and can be generally divided into chromatographic methods, for example, ion exchange chromatography, molecular weight sieving, high pressure liquid chromatography, affinity chromatography, and electrophoretic methods, for example, electrophoresis on agarose or acrylamide gels and isoelectric focusing. Any of these methods can be adapted to purify ASP.
- a preferred method of purification is affinity chromatography.
- immunoaffinity chromatography an antibody to ASP is immobilized on a chromatographic substrate, a mixture containing ASP is applied to the substrate under conditions allowing the antibody to bind ASP, the unbound material is removed by washing, and the bound ASP is eluted using, for example, high or low pH, protein denaturants or chaotropes.
- ASP may be purified by affinity chromatography using one or a combination of immobilized antibodies such as those described below covalently bound to agarose beads or bound non-covalently via a Goat-anti mouse IgM antibody to Staphylococcus aureus protein G beads.
- ASP isolation can also be achieved, for example, by incubating cell extracts with an anti-ASP antibodies, described below, attached to a solid phase, such as chemical conjugation to agarose beads. After incubation, the beads are washed, denatured and resolved on a polyacrylamide gel.
- ASP or immunogenic fragments of ASP can be formulated and packaged using methods and materials known to those skilled in the art of vaccines, examples of which are described below.
- an immunogenic fragment of a protein is a protein fragment of at least five to eight amino acids that elicits an immune response in an animal or individual.
- ASP or immunogenic fragments of ASP can be fused to one or more other proteins or amino acid sequences. Such fusions are useful for combining multiple antigens in a single vaccine.
- Simple liquid carriers such as water or a buffered saline, can be used; either alone or in combination with other carriers.
- Adjuvants For administration as a vaccine, ASP can be combined with an adjuvant, in an amount effective to enhance the immunogenic response.
- a common adjuvant widely used in humans is alum, that is, aluminum phosphate or aluminum hydroxide. Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines. Chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, and phospholipid conjugates such as those described by Goodman-Snitkoff et al., J. Immunol. 147: 410-415 (1991), and incorporated by reference herein, can also be used.
- a preferred adjuvant is Syntex Adjuvant Formulation (SAF), which is described by Allison and Byars, J. Immunol. Methods 95: 157 (1986), and Byars and Allison, Vaccine 5: 223 (1987).
- CT cholera toxin
- mucosally delivered CT functions as a powerful immunostimulant or adjuvant of both mucosal and humoral immunity. It is therefore preferred to enhance immunogenicity of the orally administered antigen by including CT in the vaccine.
- adjuvants examples include muramyl dipeptides, muramyl tripeptide, cytokines, diphtheria toxin, and exotoxin A.
- Commercially available adjuvants include QS-21 from Cambridge Biosciences, Worcester, MA, and monophosphoryl lipid A (MPLA) from Ribi Immunochem.
- the carrier may also be a polymeric delayed release system. Synthetic polymers are particularly useful in the formulation of a vaccine to effect the controlled release of antigens. An example of this is described by Kreuter, Microcapsules and Nanoparticles in Medicine and Pharmacology, pages 125-148 (M. Donbrow, ed., CRC Press). The use of other particles have demonstrated that the adjuvant effect of these polymers depends on particle size and hydrophobicity.
- Microencapsulation has been applied to the injection of microencapsulated pharmaceuticals to give a controlled release.
- a number of factors contribute to the selection of a particular polymer for microencapsulation.
- the reproducibility of polymer synthesis and the microencapsulation process, the cost of the microencapsulation materials and process, the toxicological profile, the requirements for variable release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered.
- useful polymers are polycarbonates, polyesters, polyurethanes, polyorthoesters, and polyamides, particularly those that are biodegradable.
- PLGA poly (d,l-lactide-co-glycolide)
- PLGA poly (d,l-lactide-co-glycolide)
- the PLGA microencapsulation process uses a phase separation of a water-in-oil emulsion.
- ASP is prepared as an aqueous solution and the PLGA is dissolved in a suitable organic solvents such as methylene chloride and ethyl acetate. These two immiscible solutions are co-emulsified by high-speed stirring. A non-solvent for the polymer is then added, causing precipitation of the polymer around the aqueous droplets to form embryonic microcapsules.
- microcapsules are collected, and stabilized with one of an assortment of agents (polyvinyl alcohol (PVA), gelatin, alginates, polyvinylpyrrolidone (PVP), methyl cellulose) and the solvent removed by either drying in vacuo or solvent extraction.
- agents polyvinyl alcohol (PVA), gelatin, alginates, polyvinylpyrrolidone (PVP), methyl cellulose
- Proteosome Carriers Proteosomes, combinations of protein and liposomes, can also be used as carriers for ASP; using ASP as the protein component.
- the procedures and materials for the use of proteosomes are as described in Lowell et al., Science 240:800 (1988); Lowell, in New Generation Vaccines (Woodrow and Levine, eds., Marcel Dekker, NY, 1990), Ch. 12, pages 141-160; and Orr et al., Infect. Immun. 61: 2390 (1993), the teachings of which are incorporated herein.
- the immunogenic vaccine composition can contain other physiologically acceptable ingredients such as water, saline or a mineral oil such as DrakeolTM, MarkolTM, and squalene, to form an emulsion, or in combination with aqueous buffers, or encapsulated within a capsule or enteric coating to protect the protein from degradation while passing through the stomach.
- physiologically acceptable ingredients such as water, saline or a mineral oil such as DrakeolTM, MarkolTM, and squalene
- Nucleic acid encoding all or an immunogenic part of ASP can also be used as or in a vaccine.
- a benign microorganism expressing ASP can be administered as a vaccine.
- a vaccine of this type is described by Eisinstein, Science 214: 337-338 (1981).
- Nucleic acid encoding ASP can also be directly administered, in the manner described by Liu et al., AIDS Research and Human Retroviruses 10: S34 (1994), and Donnelly et al., J. Immunol. Methods 176(2): 145-152 (1994).
- the vaccine is packaged in a single dosage for immunization by parenteral, that is, intramuscular, intradermal or subcutaneous, administration; or nasopharyngeal, that is, intranasal, administration.
- the effective dosage is determined using standard techniques, such as antibody titer.
- the antigen may be lyophilized for resuspension at the time of administration or in solution. If administered with adjuvant, the adjuvant may be administered in combination with or in the vicinity of the vaccine.
- Immunity can be measured using assays to detect and quantitate antibodies that bind to ASP.
- Cellular immunity can be measured using assays that measure specific T-cell responses such as delayed type hypersensitivity (DTH) and lymphocyte proliferation.
- DTH delayed type hypersensitivity
- the dosage is determined by the antigen loading and by standard techniques for determining dosage and schedules for administration for each antigen, based on titer of antibody elicited by the antigen administration.
- a dose effective to elicit an immune response is considered to be one that causes antibody titer to increase compared to untreated animals or individuals, using any of the known methods of titering antibodies.
- ASP vaccine use in animals and to test the use of ASP as a vaccine, the following procedure can be used.
- Dogs in the experimental group can be immunized with 0.2 mg of recombinant ASP in the presence of an equal volume of aluminum hydroxide (alum).
- the initial injection can be performed at an intramuscular (i.m.) site.
- control dogs can be injected with alum alone.
- Boosts can be administered over two week intervals.
- the first boost can also use 0.2 mg recombinant ASP injected i.m. in the presence of an alum adjuvant, while the second boost can use 0.1 mg of recombinant ASP in buffer administered by the intravenous route.
- Circulating antibodies to recombinant ASP can be detected by enzyme immunoassay using recombinant ASP as antigen. Such assays are described below. Briefly, plates can be coated with 1 microgram of recombinant ASP per well. Horse radish peroxidase (HRP)-conjugated goat anti-dog IgG antibodies is used at 1:1,000 dilution. Canine immune responses can also be measured by immunofluorescence (IFA), two-direction agarose diffusion and by Western/immunoblotting as described by Liu Shu-xian et al., SE Asian J. Trop. Med. Pub. Health 24: 61-65 (1993).
- HRP horse radish peroxidase
- IFA immunofluorescence
- Detection and quantitation of ASP, hookworm nucleic acid, and anti-ASP antibodies in clinical samples and body fluids can be accomplished using any of the known methods for detection of antigens, nucleic acids, and antibodies. Such assay are useful for determining if an individual is infected with hookworm, whether an individual is producing hookworm antibodies, and for testing the concentration or potency of ASP vaccines or ASP antibodies.
- an antibody specific for ASP protein is an antibody that is immunoreactive with an ASP protein, as defined herein, but that is not immunoreactive with hymenoptera proteins.
- mice Animals such as mice may be immunized by administration of an amount of ASP, or a fragment of ASP, effective to produce an immune response.
- a mouse is subcutaneously injected in the back with 100 micrograms of antigen, followed three weeks later with an intraperitoneal injection of 100 micrograms of ASP with adjuvant, most preferably Freund's complete adjuvant. Additional intraperitoneal injections every two weeks with adjuvant, preferably Freund's incomplete adjuvant, may be necessary until the proper titer in the mouse's blood is achieved.
- a titer of at least 1:5000 is preferred, and a titer of 1:100,000 or more is most preferred.
- Rabbits may be immunized by administration of ASP, or a fragment of ASP, effective to produce an immune response.
- a rabbit is intradermally and intravascularly injected at multiple sites with an adjuvant, preferably Freund's complete adjuvant, and ASP coupled to a carrier.
- In vitro immunization of human lymphocytes can be used to generate human monoclonal antibodies (MAb) to ASP.
- Techniques for in vitro immunization of human lymphocytes are well known to those skilled in the art. Examples of this method are described by Inai et al., Histochemistry (Germany) 99(5): 335-362 (May 1993); Mulder et al., Hum. Immunol. 36(3): 186-192 (Mar. 1993); Harada et al., J. Oral Pathol. Med. (Denmark) 22(4): 145-152 (Apr. 1993); Stauber et al., J. Immunol.
- MAbs to ASP can be produced after immunization of mice with purified ASP or a fragment of ASP.
- Hybridomas are produced using spleen cells from mice immunized with the antigen. The spleen cells of each immunized mouse is fused with mouse myeloma Sp 2/0 cells, for example using the polyethylene glycol fusion method described by Galfre and Milstein (1981). Growth of hybridomas, selection in HAT medium, cloning and screening of clones against antigens are carried out using standard methodology described by Galfre and Milstein (1981).
- HAT-selected clones are injected into mice to produce large quantities of MAb in ascites, as described by Galfreand Milstein (1981), and the MAbs in ascites may then, if desired, be purified using protein A column chromatography.
- MAbs are selected on the basis of their (a) specificity for ASP, (b) high binding affinity, (c) isotype, and (d) stability.
- ASP antibodies can be purified by affinity chromatography using antigen molecules immobilized on a solid support.
- Anti-ASP antibodies can be directly or indirectly labelled with a detectable label to facilitate detection of the presence of the antibodies by detection of the label.
- a detectable label such as, but not restricted to, 32 P, 3 H, 14 C, 35 S, 125 I, or 131 I.
- the radiolabel is generally attached by chemical modification. Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography.
- Fluorogens can also be used directly or indirectly to label the anti-ASP antibodies.
- fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, or Texas Red.
- the fluorogens are generally attached by chemical modification and can be detected by a fluorescence detector.
- the anti-ASP antibody can alternatively be labelled directly or indirectly with a chromogen to provide an enzyme or affinity label.
- the antibody can be biotinylated so that it can be utilized in a biotin-avidin reaction which may also be coupled to a label such as an enzyme or fluorogen.
- the antibody can be labelled with peroxidase, alkaline phosphatase or other enzymes giving a chromogenic or fluorogenic reaction upon addition of substrate.
- Additives such as 5-amino-2,3-dihydro-1,4-phthalazinedione, also known as LuminolTM(Sigma Chemical Company, St.
- rate enhancers such as p-hydroxybiphenyl, also known as p-phenylphenol (Sigma Chemical Company, St. Louis, Mo.) can be used to amplify enzymes such as horseradish peroxidase through a luminescent reaction; and luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used.
- enzymes such as horseradish peroxidase through a luminescent reaction
- luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used.
- Such labels can be detected using enzyme-linked immunosorbent assays (ELISA) or by detecting a color change with the aid of a spectrophotometer.
- ELISA enzyme-linked immunosorbent assays
- Anti-ASP antibodies can be screened or tested for specificity using any of a variety of standard techniques, including Western Blotting and ELISA, both described by Koren et al., Biochim. Biophys., Acta 876: 91-100 (1986). Many assays useful for determining immunoreactivity and specificity are described in Johnstone and Thorpe, Immunochemistry in Practice, Second edition (Blackwell Scientific Publication, Oxford, 1987). For example, in ELISA, separate wells in microtiter plates are coated with purified ASP which adsorb to the wall of the wells. The wells are then treated with a blocking agent, such as bovine serum albumin or nonfat milk proteins, to cover areas in the wells not bound by antigen.
- a blocking agent such as bovine serum albumin or nonfat milk proteins
- Ascites fluid or other antibody-containing preparation can then be applied to each well in varying concentrations and adequate time allowed for antibody to bind the antigen adsorbed on the wall of each well.
- the presence of antibody bound to antigen in a well can then be detected using a standard enzyme-conjugated anti-mouse antibody which will bind antibody that has bound to ASP in the well.
- Wells in which antibody is bound to antigen are then identified by adding a chromogenic substrate for the enzyme conjugated to the anti-antibody antibody and color production detected by an optical device such as ELISA plate reader.
- Antibodies that bind to a particular ASP fragment with no reactivity with other ASP regions are considered specific for that fragment.
- specificity of MAbs for a particular ASP fragment individual wells on ELISA plates are coated with various ASP fragments and subjected to the identical procedure.
- a competitive ELISA is carried out as follows. For example, one of the antibodies is biotinylated whereas the other is not so modified. Mixtures containing a constant concentration of the biotinylated antibody and increasing concentrations of the non-biotinylated antibody are incubated with wells coated with the appropriate ASP fragment.
- the quantity of biotinylated antibody bound to the coated antigen is determined using a streptavidin-peroxidase conjugate and a chromogenic substrate.
- a decreased binding of the biotinylated antibody with increasing concentrations of the non-biotinylated antibody indicates that the two antibodies compete for the same epitope. If the biotinylated antibody binds equally to the antigen despite increasing concentrations of the non-biotinylated antibody, the two antibodies do not compete for the same epitope. This competition can be complete or partial.
- Affinity of antibodies can be determined using radioactively labelled ( 125 I) ASP and purified antibodies as described previously by Koren et al., (1986).
- Solid Phase Materials Antibodies and ASP can be bound to a solid phase material for use in assays described herein.
- Various types of adsorptive materials such as nitrocellulose, ImmobilonTM, polyvinyldiene difluoride (all from BioRad, Hercules, calif.) can be used as a solid phase material in the compositions and methods described herein.
- Pieces or strips of these materials can be coated with one or more antibodies, or functional fragments thereof, directed against specific epitopes of ASP; or with ASP, for detection of anti-ASP antibodies. Such strips are referred to herein as "dipsticks".
- the strips may also be attached to one end of a longer strip of a solid support material, such as plastic, which can serve as a handle for dipping the dipstick into various solutions and samples.
- a solid support material such as plastic
- the plastic handle can also serve as a tether so that multiple dipsticks can be attached to a common support.
- Such a multi-strip design may be particularly useful in screening for different forms of ASP simultaneously.
- pieces of the solid phase material that are coated with antibody have the general dimensions of 0.5 cm ⁇ 0.5 cm and can be attached to the longer solid support strips having general dimensions of 0.5 cm ⁇ 5 cm. Such dimensions permit an accurate determination of ASP levels in as little as 100 ⁇ l of blood.
- the solid phase material may be coated with antibodies or ASP by any of a variety of methods. If the material is made of a protein-receptive solid phase material that adsorbs proteins, such as nitrocellulose or polyvinyldiene difluoride (PVDF) membrane, the material can be coated directly with antibody or ASP by immersing the solid phase material directly into a solution of the protein.
- a protein-receptive solid phase material that adsorbs proteins such as nitrocellulose or polyvinyldiene difluoride (PVDF) membrane
- the material is treated ("blocked") with a blocking agent in order to minimize nonspecific adsorption of proteins to unoccupied domains on the material.
- the solid phase material can be treated with any of a variety of blocking agents such as bovine serum albumin (BSA), gelatin, Tris, all of which are available commercially (Sigma, St Louis, Mo.) or nonfat milk proteins.
- BSA bovine serum albumin
- Tris Tris, all of which are available commercially (Sigma, St Louis, Mo.) or nonfat milk proteins.
- PVDF strips can be blocked with two percent (w/v) milk blocking solution (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) for 48 hours at 4° C.
- a dipstick may be coated with more than one antibody or ASP so that the single dipstick can be used to detect more than one anti-ASP antibody or more than one form of ASP.
- two or more separate pieces of a solid phase material, each coated with an antibody directed against a particular ASP or ASP fragment, can be attached (for example, by gluing) to a longer strip of solid support to produce a dipstick with two or more separate areas.
- ASP can be detected and quantitated using any of the known methods of protein detection. Examples include radioimmunoassays (RIA), Western Blotting (Koren et al. (1986)) and enzyme-linked immunosorbent assay (ELISA)(Koren et al. (1986)). Many assays useful for detection of ASP are described in Johnstone and Thorpe, Immunochemistry in Practice, Second edition (Blackwell Scientific Publication, Oxford, 1987). Preferred assays use the ASP-specific antibodies and antibody compositions described above. All of the antibody assays involve bringing into contact a sample to be tested and an antibody that recognizes an Ancylostoma Secreted Protein under conditions promoting conjugation of an Ancylostoma Secreted Protein and the antibody.
- RIA radioimmunoassays
- ELISA enzyme-linked immunosorbent assay
- Detection and quantitation is typically facilitated by using labelled antibody as described above.
- Detection of a radiolabel can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography.
- Fluorogens can be detected by a fluorescence detector.
- Antibodies directly or indirectly labelled with chromogens are detected visually or spectrophotometrically. Such by detection is preceded by an enzymatic development step when using some chromogens.
- Antibodies that recognize ASP can be detected and quantitated using any of the known methods of antibody detection. Examples include dot blot assays and ELISA. Detection of antibodies that recognize ASP is useful for determining if an individual has ever been infected with hookworm and to evaluate antibody preps. Detection of these antibodies involves bringing into contact a sample to be tested and ASP or an immunogenic part of ASP under conditions promoting conjugation of antibody and ASP. The bound protein is then detected, and, depending on the assay, quantitated. Detection and quantitation is typically facilitated by using labelled protein.
- ASP can be used in solution or immobilized to a solid substrate, such as a gel suitable for affinity chromatography, or a multi-well plate, using standard techniques known to those skilled in the art.
- the peptides can be labelled using standard techniques, for use in routine assays, with radioactive, fluorescent, or enzyme labels. Detection of a radiolabel can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography. Fluorogens can be detected by a fluorescence detector. Proteins directly or indirectly labelled with chromogens are detected visually or spectrophoto-metrically. Such by detection is preceded by an enzymatic development step when using some chromogens.
- An advantage of the cloned antigens is the ease of preparation of the antigen for use in ELISA.
- ELISA has advantages over other techniques for quantitation of antibody, which can be used to determine antibody titer.
- the cloned antigen can also be used to simplify detection of the antigen in a dot blot assay, which cannot be done with whole extract.
- a dot blot assay can be modified to a "dip-stick" type of test to make it even more simple and incorporate it into a test for multiple specificities at once.
- Nucleic acid molecules encoding all or a part of ASP can be performed using any of the many well known nucleic acid detection methods.
- Nucleic acid hybridization assays generally involve bringing into contact a sample to be tested and a labelled nucleic acid probe encoding ASP under conditions promoting hybridization of nucleic acid molecules. The bound nucleic acid is then detected, and, depending on the assay, quantitated.
- the detectable portion of the bound probe has traditionally been a substance chemically affixed to the probe such as a chemical label.
- chemical labels are radioisotopes, substrate-modifying enzymes such as alkaline phosphatase, enzymatic cascade processes utilizing NADP, and fluorescent compounds.
- Assays utilizing nucleic acid probes, to which a chemical label has been attached, are described by Grunstein et al., Proc. Natl. Acad. Sci. USA 72: 3961 (1975) and Southern, J. Mol. Biol. 98: 503 (1975), and in U.S. Pat. No. 4,851,331 to Vary et al.; U.S. Pat. No.
- Recent probe technology has utilized signal-amplification systems that amplify the signal for detection of smaller quantities of target molecule. For example, in nucleic acid assays, the polymerase chain reaction (PCR) and similar processes are used to increase the number of copies of the target molecule to which the detectable probe will bind.
- PCR polymerase chain reaction
- PCR has emerged as a powerful and sensitive procedure for the amplification of specific DNA sequences, and is a valuable tool in paternity identification, tissue typing, and forensics.
- PCR technology requires pairs of dissimilar DNA oligonucleotides that act as primers to initiate a controlled polymerase reaction for amplification of the DNA sequence that lies between the two oligonucleotide binding sites.
- the polymerase chain reaction employs the heat-stable Taq polymerase that permits repeated heating and cooling of the reaction mixture.
- the amplification process is initiated by first heating the reaction mixture to denature, or dissociate, the two complementary strands of the double-stranded DNA to be amplified.
- each single-stranded DNA oligonucleotide hybridizes to a specific region of one or the other of the complementary DNA strands and acts as a primer for the heat-stable polymerase.
- the polymerase uses the oligonucleotide primers as starting points for the elongation of a DNA molecule complementary to the template DNA molecule to which each primer is hybridized.
- Each of the elongating DNA chains grows towards and beyond the distal primer site of the other template strand.
- the cycle is repeated many times, exponentially doubling the number of copies each time. In this way, even a single copy of a specific DNA sequence can be amplified to detectable levels in a relatively short period of time.
- RNA polymerase promoter sequence is included as part of a single-stranded DNA probe so that, after hybridization of the probe to the target, the promoter can be used to promote polymerase-catalyzed synthesis of target or probe segments.
- Assays employing DNA transcription-based amplification systems are described by Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173-1177 (1989); Guarelli et al., Proc. Natl. Acad. Sci.
- RNA based amplification system a nucleic acid probe containing an RNA molecule is autocatalytically replicated using Q ⁇ bacteriophage replicase as described by Chu et al., Nucl. Acids Res. 14: 5591-5603 (1986); U.S. Pat. No. 4,957,858 to Chu et al.; U.S. Pat. No. 4,786,600 to Kramer et al.; and International Pat. Application No. WO92/12261 to Promega Corp.
- Probes have been indirectly labelled in an attempt to avoid the problems associated with direct radioactive labelling.
- the most common method of indirect labelling is to attach biotin, a small vitamin, to the nucleic acid using a chemical or enzymatic technique. Following hybridization, the biotin is detected by reaction with avidin, an egg white protein which has been labelled with an enzyme or fluorochrome. Bound enzyme can be detected by reaction with color-producing substrates and the fluorochrome can be seen when reacted with incident light of appropriate wavelength.
- Hybridization has been detected with the use of an intercalating agent such as acridine orange or ethidium bromide as described in U.S. Pat. No. 4,563,417 to Albarella et al.
- the intercalating agent becomes inserted between hybridized base pairs of probe and sample nucleic acids and causes the tertiary structure of the helix to unwind.
- An antibody specific for the newly formed antigenic determinant created by the intercalating agent and the unwound helix is detected by conventional means.
- Hybridization can also be detected with the aid of an antibody specific for a labelled probe as described in U.S. Pat. No. 4,743,535 to Carrico.
- the probe is labelled with a detectable substance such as flavin adenine dinucleotide (FAD) or a fluorescent agent.
- FAD flavin adenine dinucleotide
- An antibody specific for the labelled probe, after it has hybridized to the sample nucleic acid, is detected by a biochemical reaction.
- Therapeutics that can bind to, and inhibit the effect of, ASP during hookworm infection can be designed.
- Computer modeling technology allows visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule.
- the three-dimensional construct typically depends on data from x-ray crystallographic analyses or NMR imaging of the selected molecule.
- the molecular dynamics require force field data. Methods for generating this data and determining three-dimensional structure of a protein are known to those of skill in the art.
- the computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
- CHARMm performs the energy minimization and molecular dynamics functions.
- QUANTA performs the construction, graphic modelling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
- L 3 were collected from one to four week old coprocultures by the Baermann technique, and decontaminated with 1% HCl in BU buffer (50 mM Na 2 HP0 4 /22 mM KH 2 PO 4 /70 mM NaCl, pH 6.8) (Hawdon and Schad, Journal of the Helminthological Society of Washington 58: 140-142 (1991)) for 30 minutes at 22° C.
- Approximately 3500-7500 L 3 were "activated” by incubation in 0.5 mL RPMI1640 tissue culture medium (supplemented with 0.25 mM HEPES pH 7.2, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 100 ⁇ g/mL gentamicin, and 2.5 ⁇ g/mL amphotericin B) containing 15% (v/v) of a less than 10 kDa filtrate of canine serum (FIL) (Hawdon et al. (1995)) and 25 mM GSM in individual wells of 12 well tissue culture plates.
- Non-activated L 3 were incubated in RPMI alone. The larvae were incubated at 37° C., 5% CO 2 for 24 hours.
- ES products from approximately 50,000 activated A. caninum L 3 were concentrated and electrophoresed in a 12.5% acrylamide gel (Laemmli, Nature (London) 227: 680-685 (1970)) under non-reducing conditions.
- a band of molecular weight (MW) 37-40 kDa was excised, digested with trypsin in situ, and the peptides separated by HPLC.
- One peptide was subjected to sequential Edman degradation, yielding the sequence Gly-Leu-Glu-Pro-Asp-Ala-Leu-Gly-Gly-Asn-Ala-Pro-Lys (SEQ ID NO:3). Sequencing was performed by the Keck Microsequencing facility at Yale University.
- Degenerate primers in both orientations were synthesized based on the amino acid sequence, and used to amplify an ASP gene-specific product from DNA isolated from an A. caninum L 3 cDNA library constructed in lambda ZAP IITM (Stratagene).
- the degenerate primers were paired with opposing flanking vector primers (T3 or T7 promoter) in preliminary experiments.
- the sense strand degenerate primer ASP-5 SEQ ID NO: 11; Table 1
- the T7 primer SEQ ID NO: 16; Table 1 amplified a product of approximately 550 bp, and therefore these primers were used to amplify DNA for cloning.
- the reaction conditions for PCR were 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 1 U Taq DNA polymerase (Promega), and 75 ng of phage DNA containing the L 3 cDNA library in a 20 ⁇ l reaction.
- Ten identical reactions containing only the DNA and primers in 10 ⁇ l were subjected to "hot start" PCR (Amheim and Erlich, Annual Review of Biochemistry 61: 131-156 (1992)) by heating at 94° C. for 5 minutes, then lowered to 85° C. for 5 minutes, during which time the remaining reaction components were added.
- the reactions were subjected to 30 cycles of 1 minute denaturation at 94° C., 1 minute annealing at 45° C., and 2 minutes extension at 72° C. Following amplification, the products were pooled and digested with PstI and KpnI. The digested DNA was electrophoresed in a 3% low melting point NuSieveTM GTG agarose gel (FMC Bioproducts) containing 0.5 ⁇ g/mL ethidium bromide.
- a band of approximately 550 bp was excised, transferred to a microfuge tube, and melted at 65° C.
- the melted agarose was frozen in a dry ice-ethanol bath, and immediately centrifuged at 16,000 ⁇ g for 10 minutes.
- the aqueous phase was transferred to a new tube, ethanol precipitated, resuspended in 7 ⁇ L of dH 2 0, and cloned into pBluescriptTM by standard methods.
- Double-stranded plasmid DNA was sequenced by the dideoxy method (Hattori and Sakaid (1986); Sanger et al. (1977)), using the SequenaseTM 2.0 kit (USB) and sequential synthetic oligonucleotide primers.
- the A. caninum L 3 cDNA library was propagated in XLI-BLUE cells (Stratagene, La Jolla, calif.), and plated according to standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 1989)). Approximately 2 ⁇ 10 5 plaques were screened with an RNA probe made by transcription of the 550 bp PCR product, using T3 RNA polymerase (Boehringer Manheim), in the presence of ⁇ - 32 p!-UTP.
- Plasmid DNAs were isolated by standard procedures, and digested with EcoRl and XhoI to release the inserts.
- Two of the 8 clones were of different sizes, of approximately 1000 and 1200 bp.
- the 5' and 3' ends were sequenced using T3 and T7 primers, respectively.
- Both clones had poly(A) tails 3', and identical 3' sequences.
- the 5' ends were different, but further sequencing indicated that the shorter clone was a truncated version of the longer clone, renamed pASP-1.
- Restriction mapping indicated internal EcoRI and NcoI restriction sites at nucleotide positions 284 and 888 of SEQ ID NO: 1, respectively, which were utilized for subcloning to facilitate sequencing. Both strands were sequenced completely.
- a modified 5'-RACE technique was used to isolate the 5' end and start codon of the ASP cDNA (Frohman et al., Proceedings of the National Academy of Sciences USA 85: 8998-9002 (1988); Jain and Gomer, Biotechniques 12: 58-59 (1992); Templeton et al., Biotechniques 15: 48-51 (1993)).
- Approximately 400 mg of frozen A. caninum L 3 were ground to a powder in a mortar chilled in liquid nitrogen, and total RNA isolated using the TRIzol reagent (BRL).
- First strand cDNA was synthesized using M-MLV reverse transcriptase (SuperscriptTM II, BRL) and one pmole of ASP gene-specific primer 1 (GSP 1; SEQ ID NO: 12; Table 1) according to the manufacturer's protocol.
- the cDNA was ethanol precipitated in the presence of 10 ⁇ g of yeast tRNA, and resuspended in 200 ⁇ l dH 2 0.
- the cDNA was 3'-tailed with in a 50 ⁇ l reaction containing 33.5 ⁇ l cDNA, 1 mM dGTP, and 15 U of terminal deoxytransferase (TdT, BRL) for 1 hour at 37° C.
- reaction was extracted once with phenol:chloroform:isoamyl alcohol (25: 24: 1), ethanol precipitated, and resuspended in 150 ⁇ l of dH 2 0.
- Two ⁇ l were used as template for the first PCR reaction, containing 25 pmoles of GSP 2 antisense primer (SEQ ID NO: 13), located internally to the primer used for RT (Table 1), together with 25 pmoles of a 5' poly(G) anchor primer (SEQ ID NO: 15; Table 1).
- the 50 ⁇ l reaction was subjected to 40 cycles of one minute at 94° C., one minute at 48° C., and 1 minute at 72° C.
- ES products were electrophoresed in 11% polyacrylamide gel and transferred to ImmobilonTM-P PVDF membranes (Millipore) by electroblotting at 20V for 16 to 18 hours at 4° C.
- the membranes were blocked with 5% non-fat dry milk (Carnation) in PBS (NFDM) for 6 to 8 hours at 4° C.
- NFDM non-fat dry milk
- the membrane was incubated with a 1:1000 dilution of rabbit antiVespula squamosa antigen 5 (Vesq Ag5) antibody (a gift of Donald Hoffman, Dept. of Pathology, East Carolina University) in NFDM for 16-18 hours at 4° C.
- the membrane was incubated with a 1:5000 dilution of HRP-conjugated goat anti-rabbit secondary antibody (Sigma) for one hour at 22° C. Bands were visualized using Enhanced Chemiluminescence (Amersham).
- a 1 kb EcoRI/XhoI fragment containing the 3' end and poly(A) tail of ASP-1 was cloned in-frame into the pET28(c) expression vector (Novagen) and used to transform competent BL21 E. coli cells.
- Log-phase cells were induced by the addition of one mM IPTG, and incubated for three hours. One milliliter aliquots were removed after 0, 1, 2, and 3 hours of induction, and examined by SDS-PAGE and Western blotting.
- the pellet containing the expressed ASP was resuspended in 60 mL 1% SDS/0.5% 2-mercaptoethanol (2-ME), boiled for 5 minutes, and incubated at 22 C. for 14-18 hours (overnight).
- the lysate was transferred to dialysis membrane (12,000-14,000 molecular weight cut-off) and dialyzed against two liters of 0.1% SDS in PBS for two days, with two changes of buffer.
- the pET28 expression system is designed to attach a short leader containing 6 histidine residues on the amino terminus of the expressed protein. This allows purification of the recombinant protein using a Ni ++ chelating resin (HisBind, Novagen) to bind the His tag.
- Recombinant ASP was purified from the cell extract according to the manufacturers's instructions, except that 0.1% SDS was added to the column buffers. The protein was eluted from the column with 1.0 M imidazole, dialyzed as above, and examined by SDS-PAGE. Protein concentration was determined by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit, Pierce).
- the L 3 were removed, pelleted, and the supernatant transferred to a new tube and concentrated by lyophilization.
- the L 3 were returned to a new well, fresh medium containing the stimuli was added, and the incubation resumed, except at 24 hours, which was the terminal time point.
- a negative control (no stimuli) and a positive control (entire 24 hour ES output) were harvested at 24 hours.
- the second type of experiment was to determine the effect of feeding inhibitors on the release of ASP. Approximately 2000 L 3 were incubated with 0.5 mM 4,7-phenanthroline, a known feeding inhibitor, and activation stimuli for 24 hours. Following incubation, the ES products were harvested as above. The ES products from all experiments were examined by Western blotting as described above.
- A. caninum L 3 When A. caninum L 3 are stimulated to resume feeding in vitro, they release a major product with a Mr of approximately 40 kDa, referred to as the Ancylostoma Secreted Protein.
- microsequencing of a tryptic digest of this protein revealed a peptide with the sequence N-Gly-Leu-Glu-Pro-Asp-Ala-Leu-Gly-Gly-Asn-Ala-Pro-Lys (SEQ ID NO:3).
- This product was used as a probe to isolate a 1.2 kb cDNA clone encoding ASP from the A. caninum L 3 cDNA library.
- the cDNA contained a 3' poly(A) tail, but lacked a 5' initiation codon.
- the 5' end containing the ATG (Met) start codon was isolated from L 3 cDNA by 5' RACE.
- Four 5' end clones were sequenced, and found to encode untranslated leaders of variable sequence and length, but identical coding sequences that overlapped with the cDNA clone.
- caninum L 3 cDNA are depicted in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. None of the leaders contained the conserved nematode SL 1 or SL 2 (Bektesh and Hirsh, Nucleic Acids Research 16: 5692 (1988); Nilsen, Annual Review of Microbiology 47: 413-440 (1993); Spieth et al., Cell 73: 521-532 (1993)), nor could the 5' end be amplified from cDNA using an SL 1 primer and an internal gene specific primer.
- the full length cDNA encodes an open reading frame (ORF) of 424 amino acids (nucleotides 1-1272 of SEQ ID NO:1), with a predicted molecular weight of 45,735 and an isolelectric point of 7.13.
- the full length cDNA also has a 34 bp 3' untranslated region (nucleotides 1273-1306 of SEQ ID NO:1).
- the entire sequence of the isolated peptide is present in the ORF (amino acids 255 to 267 of SEQ ID NO:2), confirming that this cDNA encodes the ASP molecule.
- the amino terminal 18 amino acids are highly hydrophobic, and a potential eukaryotic signal sequence cleavage site is located between amino acids 18 and 19 (von Heijne, Nucleic Acids Research 14: 4683-4690 (1986)).
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Abstract
Description
TABLE 1 ______________________________________ Primers used for PCR and 5' RACE. ______________________________________ ASP 5 5'-GGTACTGCAGGARCCNGAYGCNYTNGG-3' GSP 1 5'-CCGACTCCATTATAGATGG-3' GSP 2 5'-CCACCAGCCGGAGAGC-3' GSP 3 5'-CTTGAATTCCAGCGAACGCGC-3' Anchor 5'-GAATCGATGGATCCTGCAGC.sub.(17) T7 promoter 5'-AATACGACTCACTATAG-3' ______________________________________ R = A or G; Y = C or T; N = A, C, T, or G. Restriction sites PstI (ASP 5) EcoRI (GSP 3), and BamHI (anchor) used for cloning are underlined.
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 16 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1320 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Anacylostoma caninum (D) DEVELOPMENTAL STAGE: Larva (ix) FEATURE: (A) NAME/KEY: terminator (B) LOCATION: 1273..1275 (ix) FEATURE: (A) NAME/KEY: sig.sub.-- peptide (B) LOCATION: 1..54 (ix) FEATURE: (A) NAME/KEY: mat.sub.-- peptide (B) LOCATION: 55..1272 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1272 (ix) FEATURE: (A) NAME/KEY: 3'UTR (B) LOCATION: 1273..1306 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGTTTTCACCTGTAATCGTCAGTGTGATTTTCACAATCGCCTTCTGCGATGCGTCTCCA60 GCAAGAGACGGCTTCGGCTGTTCAAACAGTGGGATAACTGACAAGGACCGGCAAGCATTC120 CTCGACTTCCACAACAATGCTCGTCGACGGGTTGCGAAAGGCGTTGAGGATAGCAACTCC180 GGCAAACTGAATCCAGCGAAGAACATGTACAAGCTGTCATGGGACTGTGCAATGGAACAG240 CAGCTTCAGGATGCCATTCAGTCATGCCCAAGCGCGTTCGCTGGAATTCAAGGTGTTGCG300 CAGAATGTAATGAGCTGGTCAAGCTCTGGTGGATTCCCCGATCCATCGGTAAAGATAGAA360 CAAACGCTCTCCGGCTGGTGGAGTGGTGCTAAAAAGAACGGCGTCGGCCCGGACAACAAA420 TACAACGGTGGCGGTCTCTTCGCCTTCTCTAACATGGTATACTCCGAAACGACGAAACTT480 GGCTGCGCCTACAAGGTTTGCGGCACTAAACTGGCGGTTTCGTGCATCTATAATGGAGTC540 GGGTACATCACAAATCAACCTATGTGGGAGACAGGTCAGGCTTGCAAGACAGGAGCAGAC600 TGCTCCACTTACAAGAACTCAGGCTGCGAGGATGGCCTTTGCACGAAAGGACCAGACGTA660 CCAGAAACAAACCAGCAGTGCCCCTCAAACACTGGAATGACTGATTCAGTCAGAGATACT720 TTCCTATCGGTGCACAATGAGTTCAGGTCGAGTGTTGCCCGAGGTCTGGAACCCGACGCT780 CTGGGCGGAAATGCACCAAAAGCAGCTAAAATGCTCAAGATGGTGTATGACTGTGAAGTA840 GAAGCATCGGCCATCAGACATGGAAATAAATGCGTCTATCAACATTCCCATGGCGAAGAC900 AGACCTGGACTAGGAGAAAACATCTACAAGACTAGTGTACTCAAATTCGATAAGAACAAA960 GCAGCCAAGCAGGCTTCACAACTCTGGTGGAATGAGTTAAAAGAGTTCGGCGTCGGCCCA1020 TCCAACGTCCTTACCACTGCTTTATGGAATAGACCCGGCATGCAGATTGGTCACTACACC1080 CAGATGGCATGGGACACCACCTACAAACTTGGATGTGCAGTTGTTTTCTGCAATGATTTC1140 ACATTCGGTGTTTGTCAGTATGGGCCAGGAGGCAATTACATGGGTCATGTCATCTACACT1200 ATGGGCCAGCCGTGTTCTCAGTGTTCGCCTGGTGCTACTTGCAGCGTGACCGAAGGCTTG1260 TGCAGTGCTCCTTAATCAGTTCTTAACAATGAATATCTTACAGTTGAAAAAAAAAAAAAA1320 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 424 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Ancylostoma caninum (D) DEVELOPMENTAL STAGE: Larva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: MetPheSerProValIleValSerValIlePheThrIleAlaPheCys 151015 AspAlaSerProAlaArgAspGlyPheGlyCysSerAsnSerGlyIle 202530 ThrAspLysAspArgGlnAlaPheLeuAspPheHisAsnAsnAlaArg 354045 ArgArgValAlaLysGlyValGluAspSerAsnSerGlyLysLeuAsn 505560 ProAlaLysAsnMetTyrLysLeuSerTrpAspCysAlaMetGluGln 65707580 GlnLeuGlnAspAlaIleGlnSerCysProSerAlaPheAlaGlyIle 859095 GlnGlyValAlaGlnAsnValMetSerTrpSerSerSerGlyGlyPhe 100105110 ProAspProSerValLysIleGluGlnThrLeuSerGlyTrpTrpSer 115120125 GlyAlaLysLysAsnGlyValGlyProAspAsnLysTyrAsnGlyGly 130135140 GlyLeuPheAlaPheSerAsnMetValTyrSerGluThrThrLysLeu 145150155160 GlyCysAlaTyrLysValCysGlyThrLysLeuAlaValSerCysIle 165170175 TyrAsnGlyValGlyTyrIleThrAsnGlnProMetTrpGluThrGly 180185190 GlnAlaCysLysThrGlyAlaAspCysSerThrTyrLysAsnSerGly 195200205 CysGluAspGlyLeuCysThrLysGlyProAspValProGluThrAsn 210215220 GlnGlnCysProSerAsnThrGlyMetThrAspSerValArgAspThr 225230235240 PheLeuSerValHisAsnGluPheArgSerSerValAlaArgGlyLeu 245250255 GluProAspAlaLeuGlyGlyAsnAlaProLysAlaAlaLysMetLeu 260265270 LysMetValTyrAspCysGluValGluAlaSerAlaIleArgHisGly 275280285 AsnLysCysValTyrGlnHisSerHisGlyGluAspArgProGlyLeu 290295300 GlyGluAsnIleTyrLysThrSerValLeuLysPheAspLysAsnLys 305310315320 AlaAlaLysGlnAlaSerGlnLeuTrpTrpAsnGluLeuLysGluPhe 325330335 GlyValGlyProSerAsnValLeuThrThrAlaLeuTrpAsnArgPro 340345350 GlyMetGlnIleGlyHisTyrThrGlnMetAlaTrpAspThrThrTyr 355360365 LysLeuGlyCysAlaValValPheCysAsnAspPheThrPheGlyVal 370375380 CysGlnTyrGlyProGlyGlyAsnTyrMetGlyHisValIleTyrThr 385390395400 MetGlyGlnProCysSerGlnCysSerProGlyAlaThrCysSerVal 405410415 ThrGluGlyLeuCysSerAlaPro 420 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Ancylostoma caninum (D) DEVELOPMENTAL STAGE: Larva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GlyLeuGluProAspAlaLeuGlyGlyAsnAlaProLys 1510 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Ancylostoma caninum (D) DEVELOPMENTAL STAGE: Larva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ACCGCGGCCCGCTTTTCTTGTTGTGCCGCTCTGTTATTTTCACACGGTTACATCTTATCA60 TG62 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Ancylostoma caninum (D) DEVELOPMENTAL STAGE: Larva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTAACGCTCTCTCTATTGTATCACGCCTCCCGCATTTCAATTCCCGGTTACATCTTATCA60 TG62 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Ancylostoma caninum (D) DEVELOPMENTAL STAGE: Larva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGTCCGGCATAATCCTTCACTCGACATCTTATCATG36 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Ancylostoma caninum (D) DEVELOPMENTAL STAGE: Larva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGGCTATAGGGAGCTCTTCGCGTTTCTTTCGTCATCTTATCATG44 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 203 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Dolichovespula arenaria (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AsnAsnTyrCysLysIleCysProLysGlyThrHisThrLeuCysLys 151015 TyrGlyThrSerMetLysProAsnCysGlyGlyLysIleValLysSer 202530 TyrGlyValThrAsnAspGluLysAsnGluIleValLysArgHisAsn 354045 GluPheArgGlnLysValAlaGlnGlyLeuGluThrArgGlyAsnPro 505560 GlyProGlnProProAlaLysAsnMetAsnLeuLeuValTrpAsnAsp 65707580 GluLeuAlaLysIleAlaGlnThrTrpAlaAsnGlnCysAsnPheGly 859095 HisAspGlnCysArgAsnThrAlaLysTyrProValGlyGlnAsnVal 100105110 AlaIleAlaSerThrThrGlyAsnSerTyrGlnThrMetSerTyrLeu 115120125 IleLysMetTrpGluAspGluValLysAspTyrAsnProHisLysAsp 130135140 LeuMetHisAsnAsnPheSerLysValGlyHisTyrThrGlnMetVal 145150155160 TrpGlyLysThrLysGluIleGlyCysGlySerValLysTyrIleGlu 165170175 AsnLysTrpHisThrHisTyrLeuValCysAsnTyrGlyProAlaGly 180185190 AsnTyrMetAsnGlnProValTyrGluArgLys 195200 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 204 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Vespula vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AsnAsnTyrCysLysIleLysCysLeuLysGlyGlyValHisThrAla 151015 CysLysTyrGlySerLeuLysProAsnCysGlyAsnLysValValVal 202530 SerTyrGlyLeuThrLysGlnGluLysGlnAspIleLeuLysGluHis 354045 AsnAspPheArgGlnLysIleAlaArgGlyLeuGluThrArgGlyAsn 505560 ProGlyProGlnProProAlaLysAsnMetLysAsnLeuValTrpAsn 65707580 AspGluLeuAlaTyrValAlaGlnValTrpAlaAsnGlnCysGlnTyr 859095 GlyHisAspThrCysArgAspValAlaLysTyrGlnValGlyGlnAsn 100105110 ValAlaLeuThrGlySerThrAlaAlaLysTyrAspAspProValLys 115120125 LeuValLysMetTrpGluAspGluValLysAspTyrAsnProLysLys 130135140 LysPheSerGlyAsnAspPheLeuLysThrGlyHisTyrThrGlnMet 145150155160 ValTrpAlaAsnThrLysGluValGlyCysGlySerIleLysTyrIle 165170175 GlnGluLysTrpHisLysHisTyrLeuValCysAsnTyrGlyProSer 180185190 GlyAsnPheMetAsnGluGluLeuTyrGlnThrLys 195200 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 205 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Polistes annulatis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ValAspTyrCysLysIleLysCysProSerGlyIleHisThrValCys 151015 GlnTyrGlyGluSerThrLysProSerLysAsnCysAlaGlyLysVal 202530 IleLysSerValGlyProThrGluGluGluLysLysLeuIleValSer 354045 GluHisAsnArgPheArgGlnLysValAlaGlnGlyLeuGluThrArg 505560 GlyAsnProGlyProGlnProAlaAlaSerAspMetAsnAspLeuVal 65707580 TrpAsnAspGluLeuAlaHisIleAlaGlnValTrpAlaSerGlnCys 859095 GlnPheLeuValHisAspLysCysArgAsnThrAlaLysTyrProVal 100105110 GlyGlnAsnIleAlaTyrAlaGlyGlySerAsnLeuProAspValVal 115120125 SerLeuIleLysLeuTrpGluAsnGluValLysAspPheAsnTyrAsn 130135140 ThrGlyIleThrLysGlnAsnPheAlaLysIleGlyHisTyrThrGln 145150155160 MetValTrpGlyLysThrLysGluIleGlyCysGlySerLeuLysTyr 165170175 MetGluAsnAsnMetGlnAsnHisTyrLeuIleCysAsnTyrGlyPro 180185190 AlaGlyAsnTyrLeuGlyGlnLeuProTyrThrLysLys 195200205 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGTACTGCAGGARCCNGAYGCNYTNGG27 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCGACTCCATTATAGATGG19 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CCACCAGCCGGAGAGC16 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CTTGAATTCCAGCGAACGCGC21 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GAATCGATGGATCCTGCAGCCCCCCCCCCCCCCCCC36 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AATACGACTCACTATAG17 __________________________________________________________________________
Claims (4)
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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US08/419,414 US5753787A (en) | 1995-04-10 | 1995-04-10 | Nucleic acids encoding ancylostoma secreted protein |
AU55379/96A AU5537996A (en) | 1995-04-10 | 1996-04-10 | Hookworm vaccine |
PCT/US1996/004821 WO1996032479A1 (en) | 1995-04-10 | 1996-04-10 | Hookworm vaccine |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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US08/419,414 US5753787A (en) | 1995-04-10 | 1995-04-10 | Nucleic acids encoding ancylostoma secreted protein |
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US5753787A true US5753787A (en) | 1998-05-19 |
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US08/419,414 Expired - Fee Related US5753787A (en) | 1995-04-10 | 1995-04-10 | Nucleic acids encoding ancylostoma secreted protein |
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US (1) | US5753787A (en) |
AU (1) | AU5537996A (en) |
WO (1) | WO1996032479A1 (en) |
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US20080311557A1 (en) * | 2007-06-15 | 2008-12-18 | Idexx Laboratories, Inc. | Device, kit and method for hookworm antigen capture and detection |
US20090286231A1 (en) * | 2007-06-15 | 2009-11-19 | Idexx Laboratories, Inc. | Methods, Devices, Kits and Compositions for Detecting Roundworm, Whipworm, and Hookworm |
US20090286230A1 (en) * | 2008-05-19 | 2009-11-19 | Idexx Laboratories, Inc. | Methods, Devices, Kits and Compositions for Detecting Roundworm |
US20090286228A1 (en) * | 2008-05-19 | 2009-11-19 | Idexx Laboratories, Inc. | Methods, Devices, Kits and Compositions for Detecting Roundworm |
US20090286227A1 (en) * | 2008-05-19 | 2009-11-19 | Idexx Laboratories, Inc. | Methods, Devices, Kits and Compositions for Detecting Whipworm |
US20100151500A1 (en) * | 2007-06-15 | 2010-06-17 | Idexx Laboratories, Inc. | Compositions, Devices, Kits and Methods for Detecting Hookworm |
US20110086340A1 (en) * | 2008-05-19 | 2011-04-14 | Idexx Laboratories, Inc. | Methods, devices, kits and compositions for detecting roundworm |
US8580518B2 (en) | 2008-05-19 | 2013-11-12 | Idexx Laboratories, Inc. | Methods, devices, kits and compositions for detecting roundworm |
US20180299466A1 (en) * | 2015-04-17 | 2018-10-18 | Kenneth Davin Fine | Diagnostic testing for immunologic food sensitivity |
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US20040052817A1 (en) * | 2002-09-13 | 2004-03-18 | Peter Geldhof | Ostertagia vaccine |
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US20090286227A1 (en) * | 2008-05-19 | 2009-11-19 | Idexx Laboratories, Inc. | Methods, Devices, Kits and Compositions for Detecting Whipworm |
US20110086340A1 (en) * | 2008-05-19 | 2011-04-14 | Idexx Laboratories, Inc. | Methods, devices, kits and compositions for detecting roundworm |
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AU5537996A (en) | 1996-10-30 |
WO1996032479A1 (en) | 1996-10-17 |
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